Neuroendocrine marker expression was increased by BYL719 treatment.

<p>A: BON-1, H727 and QGP-1 cells were treated with 10 μM BYL719 for 96 h and mRNA expression of Chromogranin A <i>(CHGA)</i>, <i>SSTR1</i>, <i>SSTR2</i> and <i>SSTR5</i> was analyzed by qPCR. B: BON-1, H727 and QGP-1 cells were treated with 1 μM BYL719 for 96 h and mRNA expression of <i>SSTR2</i> was analyzed by qPCR; Values are summarized from three independent experiments of three replicates per data point. * p<0.05; ** p≤0.01; *** p≤0.001.</p

Representative western blots of the investigated signaling pathways, apoptosis and cell cycle markers in BON-1, QGP-1 and H727 cells: The means, standard deviations and p-values of the western blot quantification and statistical analysis from at least three independent replicates are given in S1 Table.

<p>Representative western blots of the investigated signaling pathways, apoptosis and cell cycle markers in BON-1, QGP-1 and H727 cells: The means, standard deviations and p-values of the western blot quantification and statistical analysis from at least three independent replicates are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182852#pone.0182852.s031" target="_blank">S1 Table</a>.</p

CgA secretion of NET cells was not altered by BYL719 treatment.

<p>BON-1, H727 and QGP-1 cells were treated with vehicle, PDBu (positive control) or BYL719 (1 μM, 10 μM) for 24 h; the supernatant was collected for CgA measurements (two independent experiments with three replicates per data point).</p

Cell lines were treated with BYL719 (IC₅₀ and IC₈₅) for 72 h.

<p>Cells were stained with PI (DNA content) analysis solely included and mitosis-specific Phospho(Ser10)-Histone H3 immunostain followed by flow cytometry assessment (single cells). According to the stain, events were divided into cells of several cell cycle phases or were classified as sub-G1-events indicating cell death (FlowJo software), given in percent of all detected cells (mean ± SD) of at least four independent experiments. A: Cell cycle. B: Mitotic index. * p<0.05; ** p≤0.01; *** p≤0.001; **** p≤0.0001.</p

Assessment of apoptosis: Caspase 3/7 assay results after 24 h treatment with different doses of BYL719 are shown as mean percentage of caspase 3/7 activity referred to the untreated control (100%) ± SD: Strongest apoptosis was induced in BON-1 cells, followed by H727 cells.

<p>QGP-1 cells showed the least but still significant apoptosis. * p<0.05; ** p≤0.01; *** p≤0.001 (two independent experiments with three replicates per data point).</p

The combination therapy with BYL719 plus everolimus strongly induced SSTR2 expression in BON-1 and QGP-1 cells.

<p>Cells have been treated with 5 μM BYL719, 5 nM everolimus, or the combination of both for 48h (A) or with 1 μM BYL719, 1 nM everolimus or the combination of both for 96h (B). SSTR2 expression was analyzed by Western Blot (A) and qPCR (B). Representative western blot of two replicates; qPCR results are summarized from three independent experiments of three replicates. * p<0.05; ** p≤0.01; *** p≤0.001.</p